Tyrosine kinase-dependent activation of human NK cell functions upon triggering through CD44 receptor

We recently showed evidence of CD44-mediated enhancement of natural killer (NK) cell cytotoxic activity and induction of intracellular Ca²⁺ flux. In this study, we evaluated whether CD44 plays a stimulatory role in NK cell functions, such as cytokine production and activation antigen expression. Our results indicate that ligation of the CD44 receptor results in the induction of expression of the CD69 surface activation antigen as well as in the enhancement of phorbol ester-induced TNF-α secretion. We report also evidence for the coupling of CD44 receptor to a protein tyrosine kinase(s) pathway. CD44 engagement rapidly stimulates the tyrosine phosphorylation of several cellular substrates. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of CD44-stimulated phosphorylation, indicating that it activates tyrosine kinase(s). Furthermore, the drug also prevents CD44-mediated TNF-α production and CD69 expression. These findings indicate that protein tyrosine phosphorylation is an early and critical event in CD44-mediated activation of NK cell functions.     

Induction of CD16+ CD56bright NK cells with antitumour cytotoxicity not only from CD16- CD56bright NK Cells but also from CD16- CD56dim NK cells

The aim of this study was to examine the effect of cytokines on different subsets of NK cells, while especially focusing on CD16(-) CD56(dim) cells and CD16(-) CD56(bright) cells. When human peripheral blood mononuclear cells (PBMC) were cultured with a combination of IL-2, IL-12 and IL-15 for several days, a minor population of CD56(bright) NK cells expanded up to 15%, and also showed potent cytotoxicities against various cancer cells. Sorting experiments revealed that unconventional CD16(-) CD56(+) NK cells (CD16(-) CD56(dim) NK cells and CD16(-) CD56(bright) NK cells, both of which are less than 1% in PBMC) much more vigorously proliferated after cytokine stimulation, whereas predominant CD16(+) CD56(dim) NK cells proliferated poorly. In addition, many of the resting CD16(-) CD56(bright) NK cells developed into CD16(+) CD56(bright) NK cells, and CD16(-) CD56(dim) NK cells developed into CD16(-) CD56(bright) NK cells and also further into CD16(+) CD56(bright) NK cells by the cytokines. CSFE label experiments further substantiated the proliferation capacity of each subset and the developmental process of CD16(+) CD56(bright) NK cells. Both CD16(-) CD56(dim) NK cells and CD16(-) CD56(bright) NK cells produced large amounts of IFN-gamma and Fas-ligands. The CD16(+) CD56(bright) NK cells showed strong cytotoxicities against not only MHC class I (-) but also MHC class I (+) tumours regardless of their expression of CD94/NKG2A presumably because they expressed NKG2D as well as natural cytotoxicity receptors. The proliferation of CD16(+) CD56(bright) NK cells was also induced when PBMC were stimulated with penicillin-treated Streptococcus pyogenes, thus suggesting their role in tumour immunity and bacterial infections.     

The immune potential of decidua-resident CD16+CD56+ NK cells in human pregnancy

Human CD56⁺CD3^? NK cells can be subdivided into two different subsets according to the expression pattern of CD56 and CD16. CD56^+/brightCD16^? (CD16^?) NK cells are prominently cytokine producers with little cytotoxicity whereas CD56^+/dimCD16⁺ (CD16⁺) NK cells are efficient killers with poorer cytokine production potential. In human pregnancy, CD56⁺ decidual (d)NK cells accumulate in the maternal fetal interface to regulate placental immunity and development. Unlike peripheral blood (pb)NK cells, the majority of dNK cells are CD56 positive with limited CD16 reactivity. Our results demonstrated that in normal and pathological pregnancies, CD16⁺ dNK cells are a unique population in comparison to CD16^? dNK subset. The expression of NK activation receptors CD335, CD336, CD244 and CD314 on CD16⁺ dNK subpopulation was lower than that on CD16^? dNK cells. Upon cytokine stimulation with rhIL-12/15/18 or TGFβ blockade, the CD16⁺ dNK subset exhibited more robust response on the expression of IFNG, IL-8 and CD107a, compared to that of the CD16^? dNK subpopulation. Functions of the CD16⁺ dNK subset were shown to be independent of cellular interaction with trophoblast cells. Studies of preeclamptic patients revealed lower proportions of CD16⁺ dNK cells, suggesting potential protective roles of these cells during normal gestations.. Therefore, we suggest that the CD16⁺ dNK subset, through compensating CD16^? dNK cell function, is an indispensable component to regulate decidual immune response and to support placentation.     

Signaling function of reconstituted CD16: zeta: gamma receptor complex isoforms.

Natural killer cells express an Fc receptor for IgG (CD16) in association with disulfide-linked dimers composed of two homologous subunits: the zeta chain of the T cell antigen receptor complex and the gamma chain of the mast cell/basophil Fc receptor for IgE. The ability of zeta and gamma to transduce CD16-mediated activation signals was compared by reconstituting distinct CD16 receptor isoforms composed of various combinations of zeta- and gamma-containing dimers. Stably transformed non-hematopoietic and hematopoietic cell lines were established that expressed chimeric molecules comprising the extracellular domain of CD16 joined to the transmembrane and intracellular domains of zeta or gamma. Reconstituted CD16 receptor complexes triggered Ca2+ influx, tyrosine phosphorylation, and IL-2 production in stable transformants of the Jurkat T cell line. However, cross-linking of the CD16/gamma chimera induced a specific pattern of tyrosine phosphorylation and was more efficient at signal transduction than a CD16, zeta-zeta complex, suggesting that zeta and gamma cytoplasmic domains may be coupled to distinct tyrosine kinase pathways that differentially regulate CD16-mediated activation signals. By contrast, both CD16/zeta and CD16/gamma chimeric molecules were not functional in stable transformants of the fibroblast Chinese Hamster Ovary cell line, indicating a requirement for downstream signaling components present in hematopoietic cells. Finally, the zeta transmembrane domain appears to preferentially associate with CD16 rather than the CD3:TCR complex, suggesting that a hierarchy of molecular interactions governs NK and T cell differentiation.     

Expansion of CD16 positive and negative human NK cells in response to tumor stimulation

NK cells are innate immune lymphocytes that express a vast repertoire of germ‐line encoded receptors for target recognition. These receptors include inhibitory and activating proteins, among the latter of which is CD16, a low affinity binding Fc receptor. Here, we show that human NK cells expand in response to stimulation with various tumor cell lines. We further demonstrate that the tumor‐derived expansion of NK cells is accompanied by rapid, cell‐dependent, changes in CD16 expression levels. We show that in NK cells expanded in response to the EBV‐transformed cell line 721.221, CD16 is shed and therefore approximately half of the expanded 721.221‐derived NK‐cell population does not express CD16. We also show, in contrast, that in response to 1106mel cells, CD16 expression is maintained on the cell surface of the expanded NK cells due to an antibody‐dependent mechanism. Our results may provide a basis for the selective expansion of NK cells that may be used for tumor immunotherapy.     

Effect of B-cell receptor engagement on CD40-stimulated B cells.

Activation of human B cells in vitro either by cross-linking of surface immunoglobulins (sIg) or by triggering CD40 antigen, in the presence of interleukin-10 (IL-10) and interleukin-2 (IL-2), may result in high levels of immunoglobulin secretion in vitro. We studied the combined effects of ligation of the B-cell receptor (BCR) and CD40 [with anti-CD40 monoclonal antibody (mAb)] on B-cell proliferation and production of human immunoglobulin. For this purpose highly purified splenic B cells were cultured with various combinations of anti-CD40 and IL-10/IL-2 or IL-4 in the presence of CD32-transfected L cells. Simultaneous cross-linking of the BCR was achieved by mAb held on CD32-L cells or Staphylococcus aureus (SA). We found that dual BCR and CD40 ligation with IL-10/IL-2 leads to reduced immunoglobulin G (IgG) secretion compared with B cells stimulated with either anti-CD40 and IL-10/IL-2, or compared with B cells stimulated with SA or anti-BCR mAb and IL-10/IL-2. Dual BCR and CD40 ligation with anti-immunoglobulin mAb (anti-kappa + anti-lambda light chains) but not with SA induced a similar reduction in IgM production. The reduced immunoglobulin secretion found during dual ligation is accompanied by increased proliferation. This was independent of cytokine stimulation but SA/CD40-induced proliferation was increased in the presence of IL-10/IL-2, although not with IL-4. The combination anti-kappa and anti-lambda with anti-CD40 showed a long-term suppression of IgG and IgM production (at least 14 days), while anti-kappa or anti-lambda alone, or SA, allowed a moderate recovery of immunoglobulin production by day 14. These results suggest that simultaneous B-cell antigen receptor cross-linking and CD40 engagement via CD40L on T cells induces strong initial proliferation. This may be followed later by antibody production depending on the strength of the BCR signal and the presence of the appropriate cytokines.     

Selective expansion and engraftment of human CD16+ NK cells in NOD/SCID mice

NK cells are large granular lymphocytes that represent a critical component of the innate immunity. Investigations of human NK cell function are largely based on in vitro assays because of the lack of suitable animal models. Here we have established conditions leading to the development of human NK cells in NOD/SCID (severe combined immunodeficiency) mice receiving grafts of cord blood mononuclear cells (CBMC), and GFP-transduced HFWT inducing NK cells (GHINK-1), which have been shown to support the selective expansion of NK cells from human PBMC and CBMC in vitro. Significant numbers of CD56dimCD16+ cytotoxic and CD56-CD16+ immature NK cells appeared in peripheral blood (PB), peritoneal cavity, spleen, bone marrow and liver of the mice. The newly generated NK cells did not express activation markers such as CD25, CD69 and NKp44, the expression of which was augmented by IL-2 in vitro. The NOD/SCID mice engrafted with human NK cells exhibited antitumor activity against K562 erythroleukemia in vitro and in vivo. Thus, we succeeded in developing a CD56dimCD16+ cytotoxic NK cell populations in NOD/SCID mice closely resembling the main NK fraction in human PB and CD56-CD16+ immature NK cells. Our model provides not only information about the development and dynamics of physiological human NK cells but also an important pre-clinical system for immunotherapeutic strategies.     

Analysis of CD16+dim and CD16+bright lymphocytes--comparison of peripheral and clonal non-MHC-restricted T cells and NK cells.

The Fc gamma RIII receptor (CD16) has been described on natural killer cells and a small subset of T lymphocytes. CD16+bright lymphocytes represent the typical population of peripheral blood CD3- NK cells. In these studies in addition to CD16+bright NK cells Fc gamma RIII expressing cytotoxic T lymphocytes in peripheral blood from one healthy individual are characterized as CD16+dim non-MHC-restricted CTLs either expressing the alpha/beta (80%) or the gamma/delta T cell receptor (20%). Both CD16+ subsets are clearly distinct in their functional capacity performing NK and ADCC activity. Freshly isolated CD16+dim T cells exert higher ADCC, CD16+bright NK cells higher NK activity. They are also differentially activated by interleukin-2 since CD16+bright NK cells reveal a bright expression of the p75 IL-2 receptor beta-chain in contrast to the very low p75 expression on CD16+dim T cells. This activation leads to a gradual increase of ADCC by NK cells. Finally the CD16 expression pattern with low and bright intensity represents a stable phenotype expressed by clones generated from these different subpopulations. On a clonal level CD16+dim non-MHC-restricted T cells can be distinguished from CD16+bright NK cells by their lower capacity in NK killing, but they are equally potent in ADCC. Finally these CD3+CD16+dim clones provide the basis for studies of Fc gamma RIII and TcR interaction.     

Potential confusion of contaminating CD16+ myeloid DCs with anergic CD16+ NK cells in chimpanzees

Precise identification of NK-cell populations in humans and nonhuman primates has been confounded by imprecise phenotypic definitions. A common definition used in nonhuman primates, including chimpanzees, is CD3(-) CD8α(+) CD16(+) , and this is the dominant NK-cell phenotype in peripheral blood. However, recent data suggest that in chimpanzees a rare CD8α(-) CD16(+) population also exists. Herein, we present evidence validating the existence of this rare subset in chimpanzee peripheral blood, but also demonstrating that gating on CD3(-) CD8α(-) CD16(+) cells can inadvertently include a large number of CD16(+) myeloid DCs (mDCs). We confirmed the inclusion of mDCs in CD3(-) CD8α(-) CD16(+) gated cells by demonstrating high expression of CD11c, BDCA-1 and HLA-DR, and by the lack of expression of NKp46 and intracellular perforin. We also functionally validated the CD8α(-) NK-cell and mDC populations by mutually exclusive responsiveness to a classical NK-cell stimulus, MHC class I-deficient cells, and a prototypic mDC stimulus, poly I:C, respectively. Overall, these data demonstrate common problems with gating of NK cells that can lead to erroneous conclusions and highlight a critical need for consensus protocols for NK-cell phenotyping.     

IFN-gamma production in human NK cells through the engagement of CD8 by soluble or surface HLA class I molecules

The engagement of CD8 on NK cell surface by either surface or soluble HLA class I (sHLA-I) molecules induces synthesis and secretion of IFN-gamma. HLA-I-mediated effects were inhibited by the covering of CD8 with specific anti-CD8 monoclonal antibodies, indicating a direct interaction of HLA-I and CD8. That CD8 ligation induces IFN-gamma production was further supported by the finding that cross-linking of CD8 led to release of IFN-gamma at similar levels to those obtained with HLA-I. The sHLA-I-induced IFN-gamma production via CD8 was strongly down-regulated by the engagement of the inhibitory isoforms of either CD94/NKG2 complex by sHLA-I-non-(A,B,C,G) (putative sHLA-E) or CD158b by sHLA-I-Cw3 allele. Ligation of CD8 did not elicit, different from other activating NK cell surface molecules such as CD16 or CD69, triggering of NK cell-mediated cytolysis. Cyclosporin A, but not concanamycin A, an H+-ATPase vacuolar inhibitor which affects perforin and granzyme release, strongly reduced the sHLA-I-mediated CD8-dependent IFN-gamma production but did not affect cytolytic activity of NK cells, suggesting that different biochemical pathways are involved. Altogether, these findings indicate that CD8 engagement by sHLA-I activates a cyclosporin A-dependent pathway leading to production and secretion of IFN-gamma which may play a role in the regulation of innate immune responses in humans.     

Increased number of CD16CD56 NK cells in peripheral blood mononuclear cells after allogeneic cord blood transplantation

In the present study, we investigated subpopulations of natural killer (NK) cells and the expression of stimulatory and inhibitory NK receptors after adult blood and bone marrow transplantation (BBMT) and cord blood transplantation (CBT). There were significant increases in CD16⁺CD56^dim cell proportion and in absolute number in peripheral blood mononuclear cells (PBMC) during a period of 4–9 months after CBT compared with these in normal PBMC, cord blood (CB), and in PBMC after BBMT. Also, increased numbers of CD16⁺CD56^dim NK cells were sustained in some patients until 4 years after CBT. This CD16⁺CD56^dim cell subset after CBT exhibited decreased expression of NKG2A compared with that in CB and increased expression of NKG2C. Purified CD16⁺CD56^dim cells from patients 8–9 months after CBT exhibited significantly higher levels of cytolytic activity against K562 than did purified CD16⁺CD56^bright cells and also whole PBMC. The CD16⁺CD56^dim cell subset with a high level of cytolytic activity significantly increased after CBT, and these cells may be responsible for NK cell–mediated immunity after CBT.     

CD16 polymorphisms and NK activation induced by monoclonal antibody-coated target cells

CD16 and natural killer (NK) cells appear to play a central role in mediating the anti-tumor effects of monoclonal antibody (mAb) therapy, yet little is known about changes in NK cells that result from interaction of the NK cells with mAb-coated tumor cells under physiologic conditions. We developed a system using peripheral blood mononuclear cells (PBMCs) and either transformed B cells or breast cancer cells to assess how mAbs impact on NK cell phenotype. Rituximab, apolizumab and trastuzumab induced modulation of CD16 and upregulation of CD54 on NK cells when the appropriate target cells were present. Higher concentrations of mAb were needed to induce these changes on NK cells from subjects with the lower affinity CD16 polymorphism. Phenotypic changes were greater in NK cells from subjects with the higher affinity polymorphism even when saturating concentrations of mAb were used, demonstrating increased concentration of mAb can overcome some, but not all, of the influence CD16 polymorphisms have on NK activation. These studies provide a straightforward and easily reproducible technique to measure the ability of mAb-coated tumor cells to activate NK cells in vitro which should be particularly useful as mAbs with varying affinity for both target antigen and Fc receptor (FcR) are developed.     

Fc receptor beta subunit is required for full activation of mast cells through Fc receptor engagement

The high-affinity IgE receptor (Fc epsilonRI) and the low-affinity IgG receptor (Fc gammaRIII) on mast cells are the key molecules involved in triggering the allergic reaction. These receptors share the common beta subunit (FcRbeta) which contains an immunoreceptor tyrosine-based activation motif and transduces the signals of these receptors' aggregation. In rodents, FcRbeta is essential for the cell surface expression of the Fc epsilonRI. In humans, the FcRbeta gene was reported to be one of the candidate genes causing atopic diseases. However, the role of FcRbeta in vivo still remains ambiguous. To elucidate the functions of FcRbeta, we developed the mice lacking FcRbeta [FcRbeta(-/-)]. The FcRbeta(-/-) mice lacked the expression of the Fc epsilonRI on mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) was not induced in FcRbeta(-/-) mice as was expected. In these mice, the expression of IgG receptors on mast cells was augmented but the IgG-mediated PCA reaction was attenuated. Although with bone marrow-derived cultured mast cells from FcRbeta(-/-), adhesion to fibronectin and Ca2+ flux upon aggregation of IgG receptors were enhanced, mast cells co-cultured with 3T3 fibroblasts exhibited impaired degranulation on receptor aggregation. These observations indicate that FcRbeta accelerates the degranulation of mature mast cells via the IgG receptor in connective tissues.     

Phenotypic analysis of a resting subpopulation of human peripheral blood NK cells: the FcR gamma III (CD16) molecule and NK cell differentiation.

A subpopulation of human peripheral blood natural killer (NK) cells, defined by sedimentation at Percoll high buoyant densities (P greater than 1.0635-1.0640 g/ml) and unresponsiveness to interleukin-2 (IL-2), contained two distinct populations based on the intensity of CD16 (FcR gamma III) expression, namely CD16dim and CD16bright. This resting subpopulation of NK cells differed from the total population of peripheral blood NK cells, by containing a larger proportion of CD16dim cells, by the total absence of CD56bright CD16- cells, and by an inability to respond to high concentrations (500 U/ml) of rIL-2 despite the expression of an intermediate affinity (p70) IL-2R. Both CD16dim and CD16bright NK cells expressing high affinity IL-2R were initially generated following co-culture of resting NK cells with gamma-irradiated MM-170 cells and IL-2, but CD16bright NK cells became the dominant cell type later in culture. The CD16 molecule was not involved in the differentiation of resting NK cells since solid-phase-bound anti-CD16 monoclonal antibody neither enhanced nor inhibited NK cell generation. These studies demonstrate that the resting subpopulation of peripheral blood NK cells expresses a unique CD16 profile, that CD16 expression increases during NK cell generation, and that CD16 is not involved in the differentiation process.     

Specific functions of human NK cells.

Since natural killer (NK) cells lack both CD3/TCR molecules and surface Ig, it is generally thought that they are unable to recognize antigens. However, CD3-CD16+ cells were found to respond in MLC against irradiated allogeneic mononuclear cells and to lyse normal PHA blasts derived from the stimulating donor, but not autologous cells or cells derived from most allogeneic donors. A similar pattern was obtained with cloned NK cells, thus indicating that the ability to specifically recognize given normal allogeneic cells is a clonally distributed function. Moreover, analysis of NK clones for their ability to lyse either tumor cells or normal PHA blasts (both derived from individual cancer patients) indicated that the two phenomena are distinct. Analysis of a large number of NK clones derived from a given individual for their ability to lyse a panel of allogeneic donors allowed the identification of at least four groups of clones characterized by unique patterns of reactivity ("specificities"). We further studied the mode of inheritance of the various NK-defined specificities: all the characters "susceptibility to lysis" by NK clones (displaying one or another specificity) segregated independently, were inherited in an autosomic recessive manner and were carried by chromosome 6. The finding of clonally distributed specific functions in NK cells suggested the existence of clonally distributed receptor molecules. Along this line mAbs were raised against NK clones, and screened for their ability to trigger the immunizing clones: two mAbs (termed GL183 and EB6) were directed against a novel family of 58-kDa surface molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD226在NK细胞亚群上表达规律与功能关系的研究

目的：观察CD226分子在NK细胞亚群上的分布和其他NK细胞活化性受体和抑制性受体的共存规律，及与NK细胞功能的关系。方法：分别以IL-2或IL-15刺激PBMC和MLC细胞为模型，采用双重免疫荧光染色和流式细胞术分析，观察CD226分子在CD56^bright和CD56^dim NK细胞亚群上的表达，及与NK细胞活化性受体CD16和抑制性受体NKG2A的共存关系，同时用ELISA方法检测培养上清中IFN-γ的水平。用4小时^51Cr释放试实验检测NK细胞杀伤水平。结果：在PBMC中，CD226主要分布于CD56^dim亚群，在IL-2作用下，CD226主要分布于CD56^bright,亚群，而在IL-15作用下，NKG2A^ CD226^ 双阳性细胞明显增加。在MLC活化的NK细胞中，CD226主要分布于CD56^dim亚群，在IL-15作用下，CD226主要分布于CD56^bright亚群，IL-2和IL-15都能促进CD16^ CD226^ 和NKG2A^ CD226^ 双阳性细胞的增殖。IL-2和IL-15能明显提高PBMC培养上清中IFN-γ的水平，并能促进PBMC和MLC中NK细胞的杀伤活性。结论：CD226主要分布于活化NK细胞CD56^bright群上，其表达水平及与CD16及NKG2A共存关系可能受不同细胞因子调节并与NK细胞功能相关。     

1140919448CD200-dependent suppression of human NK activity by IVIG requires CD200 receptor type 2

Problem: IVIG prepared from plasma of stored human blood can be efficacious in improving pregnancy success in a selected subgroup of patients but RCTs using an IVIG showing inferior suppression of NK activity in vitro have been negative (J Assist Reprod Genet 2006). A significant component of NK suppression by IVIG appears to be due to CD200 released into plasma from PBL during storage at 4C. CD200 receptors (CD200R) are expressed at the fetomaternal interface prior to onset of abortion; CD200R1 mediates direct effects on gamma‐delta T cell development and suppresses alpha‐beta T cell responses in vitro, whereas CD200R2 alters DC so as to facilitate development of alpha‐beta Treg cells. Which receptor(s) mediate NK cell suppression? Methods: Purified human PBL or the CD56⁺ NK cell subset of PBL were used to lyse 51Cr‐labeled K562 cells in vitro. Different IVIG preparations were tested for suppressive ability, and suppression was blocked by either anti‐huCD200 mAb or rabbit anti‐huCD200R1 or R2 antibodies. Results: CD200‐dependent IVIG NK suppressive potency differed among IVIG types (Gammagard>Gamunex>>Gamimmune). CD200‐dependent suppression was blocked by anti‐CD200R antibody able to react with the type 2 receptor. K562 cells did not express receptor, and purified CD56⁺ NK cells were suppressed effectively without the need for non‐NK cells. Conclusions: IVIG may directly express NK cell activity via CD200 binding to CD200R2.